key: cord-0933291-77o0fagn
authors: Gómez, Juan; Melón, Santiago; Boga, José A.; Alvarez-Argüelles, Marta E.; Rojo-Alba, Susana; Leal-Negredo, Alvaro; Castello-Abietar, Cristian; Alvarez, Victoria; Cuesta-Llavona, Elías; Coto, Eliecer
title: Capillary Electrophoresis of PCR fragments with 5’-labelled primers for testing the SARS-Cov-2
date: 2020-05-21
journal: bioRxiv
DOI: 10.1101/2020.05.16.099242
sha: a50c675ee2f5dfc57132a5341268d40b3724870b
doc_id: 933291
cord_uid: 77o0fagn

Background Due to the huge demand for SARS-Cov-2 determination, alternatives to the standard qtPCR tests are potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples. Study design We isolated the naso-pharingeal RNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5’-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks. Results The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. Conclusion We describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR (lack of reactive and real-time PCR equipment) and increase the testing capacity.

PCR tests for the accurate and fast SARS-CoV-2 testing are crucial for the identification of carriers and control the spreading of COVID19. Most of these tests rely on the isolation and retro-transcription of the viral RNA from nasal, pharyngeal or pulmonary specimens followed by real-time quantitative PCR (qtPCR) with primers/probes designated from the SARS-Cov-2 sequence [1] [2] [3] [4] . Most of the labs use commercial kits that are usually not supplied on demand due to the huge global demand. This is a bottleneck in massive screening of this new coronavirus, and reinforces the necessity of developing new technical approaches to overcome these barriers. We describe a technical approach to SARS-Cov-2 testing by amplifying fragments of the viral genome with 5´-fluorescent primers followed by capillary electrophoresis in an ABI3130xl equipment. This method permits the analysis of 96 samples in approximately five hours.

Samples preparation. Our method was validated in nasal-swap samples from 30 individuals, 20 positive and 10 negative for SARS-Cov-2 testing with a standard qtPCR. 

We processed 20 SARS-Cov-2 positive and 10 negative samples. The accuracy of the viral RNA isolation and RT synthesis was validated by amplifying a fragment of the human ACTB gene (Figure 1 ). In the 20 positive samples we amplified two SARS-Cov-2 fragments that were further sequenced to determine the presence of nucleotide changes (Supplementary figures 2, 3) . We identified three patients who were Contributorship. All the authors contributed to this work by recruiting the cohorts or performing the genetic and statistical analysis.

None of the authors have competing interests related to this work.

All authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) the work, such as employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. The red peaks are from the size-marker (250-ROX).

Real time quantitative PCR with Taqman assay for the human ACTB gene. A typical amplification profile was observed for the cDNAs from nasalswaps in either viral positive (green) and negatives (red). Blue, negative control (genomic DNA in the PCR tube).

Guidelines for Laboratory Diagnosis of Coronavirus Disease

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Clinical evaluation of a SARS-CoV-2 RT-PCR assay on a fully automated system for rapid on-demand testing in the hospital setting

Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens

The establishment of reference sequence for SARS-CoV-2 and variation analysis

On the origin and continuing evolution of SARS-CoV-2

Genotyping coronavirus SARS-CoV-2: methods and implications

Genomic Analysis and Comparative Multiple Sequence of SARS-CoV2

Genetic diversity and evolution of SARS-CoV-2